The Circadian tau Mutation in Casein Kinase 1 Is Part of a Larger Domain That Can Be Mutated to Shorten Circadian Period.

Drosophila Double-time (DBT) phosphorylates the circadian protein Period (PER). The period-altering mutation tau, identified in hamster casein kinase I (CKIε) and created in Drosophila DBT, has been shown to shorten the circadian period in flies, as it does in hamsters. Since CKI often phosphorylates downstream of previously phosphorylated residues and the tau amino acid binds a negatively charged ion in X-ray crystal structures, this amino acid has been suggested to contribute to a phosphate recognition site for the substrate. Alternatively, the tau amino acid may affect a nuclear localization signal (NLS) with which it interacts. We mutated the residues that were close to or part of the phosphate recognition site or NLS. Flies expressing DBT with mutations of amino acids close to or part of either of these motifs produced a shortening of period, suggesting that a domain, including the phosphate recognition site or the NLS, can be mutated to produce the short period phenotype. Mutation of residues affecting internally placed residues produced a longer period, suggesting that a specific domain on the surface of the kinase might generate an interaction with a substrate or regulator, with short periods produced when the interaction is disrupted.

Drosophila spaghetti and doubletime link the circadian clock and light to caspases, apoptosis and tauopathy.

While circadian dysfunction and neurodegeneration are correlated, the mechanism for this is not understood. It is not known if age-dependent circadian dysfunction leads to neurodegeneration or vice-versa, and the proteins that mediate the effect remain unidentified. Here, we show that the knock-down of a regulator (spag) of the circadian kinase Dbt in circadian cells lowers Dbt levels abnormally, lengthens circadian rhythms and causes expression of activated initiator caspase (Dronc) in the optic lobes during the middle of the day or after light pulses at night. Likewise, reduced Dbt activity lengthens circadian period and causes expression of activated Dronc, and a loss-of-function mutation in Clk also leads to expression of activated Dronc in a light-dependent manner. Genetic epistasis experiments place Dbt downstream of Spag in the pathway, and Spag-dependent reductions of Dbt are shown to require the proteasome. Importantly, activated Dronc expression due to reduced Spag or Dbt activity occurs in cells that do not express the spag RNAi or dominant negative Dbt and requires PDF neuropeptide signaling from the same neurons that support behavioral rhythms. Furthermore, reduction of Dbt or Spag activity leads to Dronc-dependent Drosophila Tau cleavage and enhanced neurodegeneration produced by human Tau in a fly eye model for tauopathy. Aging flies with lowered Dbt or Spag function show markers of cell death as well as behavioral deficits and shortened lifespans, and even old wild type flies exhibit Dbt modification and activated caspase at particular times of day. These results suggest that Dbt suppresses expression of activated Dronc to prevent Tau cleavage, and that the circadian clock defects confer sensitivity to expression of activated Dronc in response to prolonged light. They establish a link between the circadian clock factors, light, cell death pathways and Tau toxicity, potentially via dysregulation of circadian neuronal remodeling in the optic lobes.

Circadian Rhythm Neuropeptides in Drosophila: Signals for Normal Circadian Function and Circadian Neurodegenerative Disease.

Circadian rhythm is a ubiquitous phenomenon in many organisms ranging from prokaryotes to eukaryotes. During more than four decades, the intrinsic and exogenous regulations of circadian rhythm have been studied. This review summarizes the core endogenous oscillation in Drosophila and then focuses on the neuropeptides, neurotransmitters and hormones that mediate its outputs and integration in Drosophila and the links between several of these (pigment dispersing factor (PDF) and insulin-like peptides) and neurodegenerative disease. These signaling molecules convey important network connectivity and signaling information for normal circadian function, but PDF and insulin-like peptides can also convey signals that lead to apoptosis, enhanced neurodegeneration and cognitive decline in flies carrying circadian mutations or in a senescent state.

Caspase-3-Dependent Proteolytic Cleavage of Tau Causes Neurofibrillary Tangles and Results in Cognitive Impairment During Normal Aging.

Mouse models of neurodegenerative diseases such as Alzheimer's disease (AD) are important for understanding how pathological signaling cascades change neural circuitry and with time interrupt cognitive function. Here, we introduce a non-genetic preclinical model for aging and show that it exhibits cleaved tau protein, active caspases and neurofibrillary tangles, hallmarks of AD, causing behavioral deficits measuring cognitive impairment. To our knowledge this is the first report of a non-transgenic, non-interventional mouse model displaying structural, functional and molecular aging deficits associated with AD and other tauopathies in humans with potentially high impact on both new basic research into pathogenic mechanisms and new translational research efforts. Tau aggregation is a hallmark of tauopathies, including AD. Recent studies have indicated that cleavage of tau plays an important role in both tau aggregation and disease. In this study we use wild type mice as a model for normal aging and resulting age-related cognitive impairment. We provide evidence that aged mice have increased levels of activated caspases, which significantly correlates with increased levels of truncated tau and formation of neurofibrillary tangles. In addition, cognitive decline was significantly correlated with increased levels of caspase activity and tau truncated by caspase-3. Experimentally induced inhibition of caspases prevented this proteolytic cleavage of tau and the associated formation of neurofibrillary tangles. Our study shows the strength of using a non-transgenic model to study structure, function and molecular mechanisms in aging and age related diseases of the brain.

Circadian rhythms in the Drosophila eye may regulate adaptation of vision to light intensity.

The sensitivity of the eye at night would lead to complete saturation of the eye during the day. Therefore, the sensitivity of the eye must be down-regulated during the day to maintain visual acuity. In the Drosophila eye, the opening of TRP and TRPL channels leads to an influx of Ca++ that triggers down-regulation of further responses to light, including the movement of the TRPL channel and Gα proteins out of signaling complexes found in actin-mediated microvillar extensions of the photoreceptor cells (the rhabdomere). The eye also exhibits a light entrained-circadian rhythm, and we have recently observed that one component of this rhythm (BDBT) becomes undetectable by antibodies after exposure to light even though immunoblot analyses still detect it in the eye. BDBT is necessary for normal circadian rhythms, and in several circadian and visual mutants this eye-specific oscillation of detection is lost. Many phototransduction signaling proteins (e.g., Rhodopsin, TRP channels and Gα) also become undetectable shortly after light exposure, most likely due to a light-induced compaction of the rhabdomeric microvilli. The circadian protein BDBT might be involved in light-induced changes in the rhabdomere, and if so this could indicate that circadian clocks contribute to the daily adaptations of the eye to light. Likewise, circadian oscillations of clock proteins are observed in photoreceptors of the mammalian eye and produce a circadian oscillation in the ERG. Disruption of circadian rhythms in the eyes of mammals causes neurodegeneration in the eye, demonstrating the importance of the rhythms for normal eye function.

Visual and circadian regulation of Drosophila BDBT and BDBT effects on DBT and PER localization.

BRIDE OF DOUBLETIME (BDBT) interacts with the circadian kinase DOUBLETIME (DBT) and accumulates in eye foci during the dark of a light:dark cycle. BDBT foci are shown here to be broadly expressed in constant dark and low in constant light. Analysis of circadian photoreceptor cry and visual photoreceptor ninaE mutants showed that disappearance of eye BDBT foci requires both the CRYPTOCHROME and the RHODOPSIN-1 pathways. The arr1 and arr2 mutants, which affect rhodopsin quenching, eliminated BDBT foci under dark conditions. arr1 and arr2 mutants also caused increased nuclear PER protein. The changes in BDBT foci do not result from altered BDBT levels in the eye but from changes in its immunodetection. Knockdown of BDBT specifically in the eye produced constitutively nuclear PER and constitutively cytosolic DBT. The results show that BDBT is necessary for co-transport of DBT and PER into the nucleus and suggest that this process is regulated by a light-dependent mechanism.

Drosophila and vertebrate casein kinase Idelta exhibits evolutionary conservation of circadian function.

Mutations lowering the kinase activity of Drosophila Doubletime (DBT) and vertebrate casein kinase Iepsilon/delta (CKIepsilon/delta) produce long-period, short-period, and arrhythmic circadian rhythms. Since most ckI short-period mutants have been isolated in mammals, while the long-period mutants have been found mostly in Drosophila, lowered kinase activity may have opposite consequences in flies and vertebrates, because of differences between the kinases or their circadian mechanisms. However, the results of this article establish that the Drosophila dbt mutations have similar effects on period (PER) protein phosphorylation by the fly and vertebrate enzymes in vitro and that Drosophila DBT has an inhibitory C-terminal domain and exhibits autophosphorylation, as does vertebrate CKIepsilon/delta. Moreover, expression of either Drosophila DBT or the vertebrate CKIdelta kinase carrying the Drosophila dbt(S) or vertebrate tau mutations in all circadian cells leads to short-period circadian rhythms. By contrast, vertebrate CKIdelta carrying the dbt(L) mutation does not lengthen circadian rhythms, while Drosophila DBT(L) does. Different effects of the dbt(S) and tau mutations on the oscillations of PER phosphorylation suggest that the mutations shorten the circadian period differently. The results demonstrate a high degree of evolutionary conservation of fly and vertebrate CKIdelta and of the functions affected by their period-shortening mutations.

Entrainment of the Drosophila circadian clock: more heat than light.

Circadian rhythms are produced by a biological clock that is synchronized (or entrained) by cycles of light and temperature. In Drosophila, light triggers the interaction of the photoreceptor cryptochrome (CRY) with the circadian clock protein timeless (TIM). The absence of this interaction in cryb mutants eliminates this entrainment mechanism. The abundance of TIM and period (PER) oscillate throughout the day, and they form a complex that moves to the nucleus to rhythmically repress transcription of the per and tim genes. Because the CRY:TIM interaction triggers rapid degradation of TIM, the phase of these molecular oscillations is reset by light, which thereby entrains the circadian clock. A study now shows that heat pulses trigger an association between CRY and PER:TIM, which suggests that CRY:PER:TIM also contributes to entrainment by temperature. In wild-type flies, CRY:PER:TIM formation requires high temperatures and is only triggered by heat pulses in the early night, but in per(L) mutants, which exhibit a temperature-sensitive lengthening of circadian periods, CRY:PER(L:)TIM formation is triggered by lower temperatures and throughout the night. Because CRY:PER:TIM is formed under the same conditions that entrain circadian behavior, formation of the complex is likely to mediate entrainment by heat pulses. Whereas per(L )flies exhibit longer periods at higher temperatures, per(L);cry(b) flies exhibit similar periods at different temperatures, which suggests that an altered interaction between CRY and PER(L):TIM contributes to a lack of temperature compensation. Future work should determine how the interaction between CRY and PER:TIM entrains rhythms to temperature and affects temperature compensation.

Reflections on contributing to "big discoveries" about the fly clock: Our fortunate paths as post-docs with 2017 Nobel laureates Jeff Hall, Michael Rosbash, and Mike Young.

In the early 1980s Jeff Hall and Michael Rosbash at Brandeis University and Mike Young at Rockefeller University set out to isolate the period (per) gene, which was recovered in a revolutionary genetic screen by Ron Konopka and Seymour Benzer for mutants that altered circadian behavioral rhythms. Over the next 15 years the Hall, Rosbash and Young labs made a series of groundbreaking discoveries that defined the molecular timekeeping mechanism and formed the basis for them being awarded the 2017 Nobel Prize in Physiology or Medicine. Here the authors recount their experiences as post-docs in the Hall, Rosbash and Young labs from the mid-1980s to the mid-1990s, and provide a perspective of how basic research conducted on a simple model system during that era profoundly influenced the direction of the clocks field and established novel approaches that are now standard operating procedure for studying complex behavior.

Drosophila doubletime mutations which either shorten or lengthen the period of circadian rhythms decrease the protein kinase activity of casein kinase I.

In both mammals and fruit flies, casein kinase I has been shown to regulate the circadian phosphorylation of the period protein (PER). This phosphorylation regulates the timing of PER's nuclear accumulation and decline, and it is necessary for the generation of circadian rhythms. In Drosophila melanogaster, mutations affecting a casein kinase I (CKI) ortholog called doubletime (dbt) can produce short or long periods. The effects of both a short-period (dbt(S)) and long-period (dbt(L)) mutation on DBT expression and biochemistry were analyzed. Immunoblot analysis of DBT in fly heads showed that both the dbt(S) and dbt(L) mutants express DBT at constant levels throughout the day. Glutathione S-transferase pull-down assays and coimmunoprecipitation of DBT and PER showed that wild-type DBT, DBT(S), and DBT(L) proteins can bind to PER equivalently and that these interactions are mediated by the evolutionarily conserved N-terminal part of DBT. However, both the dbt(S) and dbt(L) mutations reduced the CKI-7-sensitive kinase activity of an orthologous Xenopus laevis CKIdelta expressed in Escherichia coli. Moreover, expression of DBT in Drosophila S2 cells produced a CKI-7-sensitive kinase activity which was reduced by both the dbt(S) and dbt(L) mutations. Thus, lowered enzyme activity is associated with both short-period and long-period phenotypes.

Bone muscle crosstalk targets muscle regeneration pathway regulated by core circadian transcriptional repressors DEC1 and DEC2.

Deletion of proprotein convertase Mbtps1 in bone osteocytes leads to a significant postnatal increase in skeletal muscle size and contractile function, while causing only a 25% increase in stiffness in long bones. Concerns about leakiness in skeletal muscle were discounted since Cre recombinase expression does not account for our findings, and, Mbtps1 protein and mRNA is not deleted. Interestingly, the response of normal skeletal muscle to exercise and the regenerative response of skeletal muscle to the deletion of Mbtps1 in bone share some key regulatory features including a preference for slow twitch muscle fibers. In addition, transcriptional regulators PPAR, PGC-1α, LXR, and repressors DEC1 and DEC2 all occupy central positions within these two pathways. We hypothesize that the age-dependent muscle phenotype in Dmp1-Cre Mbtps1 cKO mice is due to bone→muscle crosstalk. Many of the myogenic genes altered in this larger and functionally improved muscle are regulated by circadian core transcriptional repressors DEC1 and DEC2, and furthermore, display a temporal coordination with Dec1 and Dec2 expression consistent with a regulatory co-dependency. These considerations lead us to propose that Dmp1-Cre Mbtps1 cKO osteocytes activate myogenesis by increased release of an activator of muscle PPAR-gamma, for example, PGE2 or sphingosine-1-P, or, by diminished release of an inhibitor of LXR, for example, long-chain polyunsaturated fatty acids. We hope that further investigation of these interacting pathways in the Dmp1-Cre Mbtps1 cKO model will lead to clinically translatable findings applicable to age-related sarcopenia and other muscle wasting syndromes.

The circadian clock-containing photoreceptor cells in Xenopus laevis express several isoforms of casein kinase I.

The frog (Xenopus laevis) retina has been an important model for the analysis of retinal circadian rhythms. In this paper, several isoforms of X. laevis casein kinase I (CKI) were analyzed to address whether they are involved in the phosphorylation and degradation of period protein (PER), as they are in the circadian oscillators of other species. cDNAs encoding two splice variants of CKI(delta) (a full-length form and deletion isoform, which is missing an exon that encodes a putative nuclear localization signal and two evolutionarily conserved protein kinase domains) were isolated and analyzed, together with a previously isolated CKI(epsilon) isoform. Both CKI(delta) and CKI(epsilon) were shown to be constitutively expressed in the photoreceptors of the retina, where a circadian clock has been localized. Both the full-length CKI(delta) and CKI(epsilon) were shown to have kinase activity in vitro, and the full-length CKI(delta) phosphorylated and degraded Drosophila PER when expressed in Drosophila S2 cells. The expression and biochemical characteristics of these CKIs are consistent with an evolutionarily conserved role for CKI in the Xenopus retinal clock. The CKI(delta) deletion isoform did not exhibit kinase activity and did not trigger degradation of PER. Subcellular localization of both CKI(delta) isoforms was cytoplasmic in several cell culture lines, but the full-length CKI(delta) , and not the deletion CKI(delta) isoform, was localized to both the nucleus and the cytoplasm in Drosophila S2 cells. These results indicate that the sequences missing in the deletion CKI(delta) isoform are important for the nuclear localization and kinase activity of the full-length isoform and that one or both of these features are necessary for degradation of Drosophila PER.

GABAB receptor subunit mRNAs are differentially regulated in pituitary melanotropes during development and detection of functioning receptors coincides with completion of innervation.

This study examines the developmental expression of GABAB receptor subunits (GABAB(1a), GABAB(1b), GABAB(2)) in the pituitary intermediate lobe using in situ hybridization, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and Western blots. Receptor functionality was studied by baclofen-stimulated GTPgammaS binding. In the adult rat pituitary all three transcripts were detected in melanotropes, but not in glia, of the intermediate lobe. No transcripts of any subunit were detected in the neural lobe. Transcripts of GABAB(1a) and GABAB(1b), but not of GABAB(2), were detected in specific subpopulations of cells in the anterior lobe. All three transcripts were detected in melanotropes on gestational day 18 using in situ hybridization. Reverse transcriptase-polymerase chain reactions comparing postnatal day 2 and adult transcript levels in the neurointermediate lobe support in situ hybridization data that GABAB(1a) mRNA levels do not change, GABAB(1b) levels increase, and GABAB(2) levels decrease as the rat matures. Thus, GABAB receptor subunit transcripts are differentially regulated in melanotropes during development. In the adult rat both GABAB(1) and GABAB(2) proteins were detected in the neurointermediate lobe using Western blotting and in melanotropes by immunohistochemistry. Developmentally, GABAB(1) protein was not detected until postnatal day 7, but was clearly expressed by postnatal day 15 while GABAB(2) protein could not be detected until postnatal day 15. Functional receptors were found in the intermediate lobe at postnatal day 15 and in the adult. The demonstration of transcripts for GABAB(1a), GABAB(1b) and GABAB(2) subunits at gestational day 18 contrasted with the failure to detect any protein before postnatal day 7, suggesting that the regulation of GABAB subunit isoforms occurs differentially at both the transcriptional and translational level as development progresses. The disparity in the regulation of the receptor subunits may suggest that GABAB(1) could have other functions besides being part of the GABAB receptor heterodimer.

A Doubletime Nuclear Localization Signal Mediates an Interaction with Bride of Doubletime to Promote Circadian Function.

Doubletime (DBT) has an essential circadian role in Drosophila melanogaster because it phosphorylates Period (PER). To determine if DBT antagonism can produce distinct effects in the cytosol and nucleus, forms of a dominant negative DBT(K/R) with these 2 alternative localizations were produced. DBT has a putative nuclear localization signal (NLS), and mutation of this signal confers cytosolic localization of DBT in the lateral neurons of Drosophila clock cells in the brain. By contrast, addition of a strong NLS domain (e.g., SV40 NLS) to DBT's C terminus leads to more nuclear localization. Expression of DBT(K/R) with the mutated NLS (DBT(K/R) NLS(-)) using a timGAL4 driver does not alter the circadian period of locomotor activity, and the daily oscillations of PER detected by immunoblot and immunofluorescence persist, like those of wild-type flies. By contrast, expression of DBT(K/R) with the strong NLS (DBT(K/R) stNLS) using the timGAL4 driver lengthens period more strongly than DBT(K/R), with damped oscillations of PER phosphorylation and localization. Both DBT(K/R) and DBT(WT) without the NLS fail to interact with Bride of Doubletime (BDBT) protein, which is related to FK506-binding proteins and shown to interact with DBT to enhance its circadian function. This result suggests that the DBT(K/R) NLS(-) has lost its dominant negative property because it does not form normal clock protein complexes. DBT(WT) proteins with the same changes (NLS(-) and stNLS) also produce equivalent changes in localization that do not produce opposite period phenotypes. Additionally, a DBT(K/R) protein with both the stNLS and NLS(-) mutation does not affect circadian period, although it is nuclear, demonstrating that the lack of a dominant negative for the DBT(K/R) NLS(-) is not due to failure to localize to nuclei. Finally, bdbt RNAi increases the cytosolic localization of DBT(K/R) but not of DBT(WT), suggesting a role for BDBT in DBT kinase-dependent nuclear localization of DBT.

The role of casein kinase I in the Drosophila circadian clock.

The circadian clock mechanism in organisms as diverse as cyanobacteria and humans involves both transcriptional and posttranslational regulation of key clock components. One of the roles for the posttranslational regulation is to time the degradation of the targeted clock proteins, so that their oscillation profiles are out of phase with respect to those of the mRNAs from which they are translated. In Drosophila, the circadian transcriptional regulator PERIOD (PER) is targeted for degradation by a kinase (DOUBLETIME or DBT) orthologous to mammalian kinases (CKIɛ and CKIδ) that also target mammalian PER. Since these kinases are not regulated by second messengers, the mechanism (if any) for their regulation is not known. We are investigating the possibility that regulation of DBT is conferred by other proteins that associate with DBT and PER. In this chapter, the methods we are employing to identify and analyze these factors are discussed. These methods include expression of wild type and mutant proteins with the GAL4/UAS binary expression approach, analysis of DBT in Drosophila S2 cells, in vitro kinase assays with DBT isolated from S2 cells, and proteomic analysis of DBT-containing complexes and of DBT phosphorylation with mass spectrometry. The work has led to the discovery of a previously unrecognized circadian rhythm component (Bride of DBT, a noncanonical FK506-binding protein) and the mapping of autophosphorylation sites within the DBT C-terminal domain with potential regulatory roles.

Drosophila DBT Autophosphorylation of Its C-Terminal Domain Antagonized by SPAG and Involved in UV-Induced Apoptosis.

Drosophila DBT and vertebrate CKIε/δ phosphorylate the period protein (PER) to produce circadian rhythms. While the C termini of these orthologs are not conserved in amino acid sequence, they inhibit activity and become autophosphorylated in the fly and vertebrate kinases. Here, sites of C-terminal autophosphorylation were identified by mass spectrometry and analysis of DBT truncations. Mutation of 6 serines and threonines in the C terminus (DBT(C/ala)) prevented autophosphorylation-dependent DBT turnover and electrophoretic mobility shifts in S2 cells. Unlike the effect of autophosphorylation on CKIδ, DBT autophosphorylation in S2 cells did not reduce its in vitro activity. Moreover, overexpression of DBT(C/ala) did not affect circadian behavior differently from wild-type DBT (DBT(WT)), and neither exhibited daily electrophoretic mobility shifts, suggesting that DBT autophosphorylation is not required for clock function. While DBT(WT) protected S2 cells and larvae from UV-induced apoptosis and was phosphorylated and degraded by the proteasome, DBT(C/ala) did not protect and was not degraded. Finally, we show that the HSP-90 cochaperone spaghetti protein (SPAG) antagonizes DBT autophosphorylation in S2 cells. These results suggest that DBT autophosphorylation regulates cell death and suggest a potential mechanism by which the circadian clock might affect apoptosis.

Regulation of sleep by neuropeptide Y-like system in Drosophila melanogaster.

Sleep is important for maintenance of normal physiology in animals. In mammals, neuropeptide Y (NPY), a homolog of Drosophila neuropeptide F (NPF), is involved in sleep regulation, with different effects in human and rat. However, the function of NPF on sleep in Drosophila melanogaster has not yet been described. In this study, we investigated the effects of NPF and its receptor-neuropeptide F receptor (NPFR1) on Drosophila sleep. Male flies over-expressing NPF or NPFR1 exhibited increased sleep during the nighttime. Further analysis demonstrated that sleep episode duration during nighttime was greatly increased and sleep latency was significantly reduced, indicating that NPF and NPFR1 promote sleep quality, and their action on sleep is not because of an impact of the NPF signal system on development. Moreover, the homeostatic regulation of flies after sleep deprivation was disrupted by altered NPF signaling, since sleep deprivation decreased transcription of NPF in control flies, and there were less sleep loss during sleep deprivation and less sleep gain after sleep deprivation in flies overexpressing NPF and NPFR1 than in control flies, suggesting that NPF system auto-regulation plays an important role in sleep homeostasis. However, these effects did not occur in females, suggesting a sex-dependent regulatory function in sleep for NPF and NPFR1. NPF in D1 brain neurons showed male-specific expression, providing the cellular locus for male-specific regulation of sleep by NPF and NPFR1. This study brings a new understanding into sleep studies of a sexually dimorphic regulatory mode in female and male flies.

Noncanonical FK506-binding protein BDBT binds DBT to enhance its circadian function and forms foci at night.

The kinase DOUBLETIME is a master regulator of the Drosophila circadian clock, yet the mechanisms regulating its activity remain unclear. A proteomic analysis of DOUBLETIME interactors led to the identification of an unstudied protein designated CG17282. RNAi-mediated knockdown of CG17282 produced behavioral arrhythmicity and long periods and high levels of hypophosphorylated nuclear PERIOD and phosphorylated DOUBLETIME. Overexpression of DOUBLETIME in flies suppresses these phenotypes and overexpression of CG17282 in S2 cells enhances DOUBLETIME-dependent PERIOD degradation, indicating that CG17282 stimulates DOUBLETIME's circadian function. In photoreceptors, CG17282 accumulates rhythmically in PERIOD- and DOUBLETIME-dependent cytosolic foci. Finally, structural analyses demonstrated CG17282 is a noncanonical FK506-binding protein with an inactive peptide prolyl-isomerase domain that binds DOUBLETIME and tetratricopeptide repeats that may promote assembly of larger protein complexes. We have named CG17282 BRIDE OF DOUBLETIME and established it as a mediator of DOUBLETIME's effects on PERIOD, most likely in cytosolic foci that regulate PERIOD nuclear accumulation.

DOUBLETIME plays a noncatalytic role to mediate CLOCK phosphorylation and repress CLOCK-dependent transcription within the Drosophila circadian clock.

Circadian clocks keep time via gene expression feedback loops that are controlled by time-of-day-specific changes in the synthesis, activity, and degradation of transcription factors. Within the Drosophila melanogaster circadian clock, DOUBLETIME (DBT) kinase is necessary for the phosphorylation of PERIOD (PER), a transcriptional repressor, and CLOCK (CLK), a transcriptional activator, as CLK-dependent transcription is being repressed. PER- and DBT-containing protein complexes feed back to repress CLK-dependent transcription, but how DBT promotes PER and CLK phosphorylation and how PER and CLK phosphorylation contributes to transcriptional repression have not been defined. Here, we show that DBT catalytic activity is not required for CLK phosphorylation or transcriptional repression and that PER phosphorylation is dispensable for repressing CLK-dependent transcription. These results support a model in which DBT plays a novel noncatalytic role in recruiting additional kinases that phosphorylate CLK, thereby repressing transcription. A similar mechanism likely operates in mammals, given the conserved activities of PER, DBT, and CLK orthologs.